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Manuscript_af48ba01d2213271109c954ef3483300

A quaternized chitin derivatives, egg white protein and montmorillonite

composite sponge with antibacterial and hemostatic effect for promoting

wound healing

Yiwen Yanga,1, Hao Zhangb,1, Fanwei Zengb, Qiaoqiao Jiaa, Lina Zhanga, Aixi Yu b,* ,

Bo Duana,*

a
College of Chemistry and Molecular Sciences, Hubei Engineering Center of Natural

Polymer-based Medical Materials, Key Laboratory of Biomedical Polymers of

Ministry of Education, Wuhan University, Wuhan 430072, China.;

b
Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, China;

* Corresponding authors.
1
These authors contributed equally to this work.

* To whom correspondence should be addressed: E-mail: bo_duan@whu.edu.cn

© 2022 published by Elsevier. This manuscript is made available under the Elsevier user license
https://www.elsevier.com/open-access/userlicense/1.0/
Abstract: In this study, we prepared an all-natural substances based sponge (EMQS)

consist of egg white protein (EW), quaternized chitin derivatives (QCs) and

montmorillonite (MMT). Inspired by the cuisine method of pastry, EW was separated

from yolk and mechanically stirred vigorously to generate fluffy and porous foam.

The incorporation of MMT enhanced the mechanical properties (a compressive

strength of 0.126 MPa), as well as the hemostatic effect of the sponge (hemostasis

time of 49.7 ± 8.0 s in a liver injury model). The QCs improved the antimicrobial

activities of EMQS against Escherichia coli (E.Coli) and Methicillin-resistant

Staphylococcus aureus (MRSA). The 3D porous structure of EMQS, coming with

beneficial properties including high porosity (84.8%), water uptake ability of 675.3

g/g and water vapor transmission rate satisfied the requirements for

soft-tissue-engineering applications. QCs interacted with MMT via hydrogen bonding

and electronical interaction so that it served as a sustainable drug release system at

wound site with excellent antibacterial activity. Three full-thickness skin defect

models of BALB/c mice indicated that this sponge could effectively shorten the

healing time and accelerate the angiogenesis and collagen deposition of uninfected,

E.Coli and MRSA infected wounds in comparison with commercial dressings. Hence,

this all-natural substances based sponge with antibacterial property and hemostatic

effect had a great potential as a clinical dressing for skin regeneration.

Key words: wound dressing; egg white protein; antibacteria; hemostat; quaternized

chitin derivatives

2
Introduction:

Skin protects the human body from thermal, chemical, and/or microbial infection

[1]. Skin defects may lead to pain, fluid loss and even life threatening complications

[2]. Wound dressing is an effective strategy for accelerating the skin defect healing by

serving as a temporary physical-chemical barrier against ambient hazard [3]. A

qualified wound dressing should exhibit certain properties such as non-toxicity,

biocompatibility, low antigenicity, good water vapor transmission rate (WVTR), and

mechanical properties [3]. Moreover, severe wounds accompanied with acute

hemorrhage and bacterial infection demand multifunctional wound dressings to

accelerate healing process. Therefore, series of advanced wound dressings have been

developed with satisfying properties of reducing blood loss and wound disinfection

[4-8]. Generally, these wound dressings were composed of several ingredients with

different functions that were combined in a designed way to ensure the maximal

promotive effect. And the diverse formations of these dressings including hydrogel [9],

electrospun mat [10] and sponge [11] have been fabricated with various functions

such as maintaining moisture, absorbing excessive exudates and allowing oxygen

permeation.

In recent years, natural polysaccharides including hyaluronic acid, alginate,

cellulose, chitin and chitosan have been widely applied for constructing tissue

engineering scaffolds due to their inherent biological applicability and structural

versatility [12, 13]. And the derivatization such as quaternarization, methacrylation

via the abundant hydroxyl and amino groups of polysaccharides has been widely

3
adopted to endow diverse functionalities to these natural polysaccharides such as

antibacterial [14], photo-crosslinking ability [15], self-healing [16], and adhesiveness

[17], to further feed the specific demands for application. Cai et al successfully

developed a one-pot and scalable synthetic approach for preparing quaternized chitin

derivatives (QCs) without toxic solvents [18]. The positively charged quaternary

ammonium groups of QCs could interact with the negatively charged bacterial cell

membranes, leading to the leakage of intracellular constituents and death of bacteria

[19, 20]. This QC has been proved to be a broad-spectrum antibacterial polymer even

to some drug resisted bacteria, letable to significantly accelerate the healing process

of Gram-positive and negative bacterial infected wounds [18, 20, 21]. Therefore, the

QCs would be the perfect candidate for the preparation of antibacterial wound

dressing.

On the other hand, egg white protein (EW) is a bio-safe, cheap, food derived

protein which has been applied as a regenerative medicine in the scope of tissue

engineering [22]. EW consists of different kinds of globular proteins including

ovalbumin (54%), ovotransferrin (12-13%), ovomucoid (11%) and lysozyme

(3.4-3.5%) [23]. EW has excellent capacity of reducing anti-inflammatory reaction

[24] and promoting the cell adhesion, proliferation and migration [25]. Moreover, the

EW based materials could significantly improve the angiogenesis and host tissue

ingrowth [26]. Besides the beneficial biological properties of EW, the physical and

chemical properties such as gelling, foaming, emulsification, heat setting and binding

adhesion [27, 28] make EW a versatile candidate for the diverse scaffolds fabrication.

4
One intriguing property of EW is the transformation from liquid into foam under

certain conditions such as mechanical stirring [29], which is a common cuisine

method to prepare spongy pastry. Inspired by this, we converted liquid EW into foam

for obtaining a porous morphology to improve the healing efficiency. Especially, 3D

porous architecture exhibits good water uptake and gas exchange ability which was

beneficial for wound exudates adsorption [30], providing a more ascendant

environment allowing related cells to proliferate and migrate unimpededly [31].

However, one disadvantage of the spongy dressing is the fragility due to the porous

structure, might be difficult to maintain its integrity at wound sites. The incorporation

of layered nanoparticles as an inorganic reinforcement nano-building block, including

montmorillonite (MMT), is a common method to improve the mechanical properties

[32]. And such layered silicates have been widely proved to be a biocompatible

material, showing no cytotoxicity and good biodegradability [33]. Moreover, as the

most effective hemostat in natural silicates, MMT could enhance the hemostatic effect

of dressings owing to its highly efficient activation of the coagulation system due to

its electronegativity [34]. And this negatively charged nanoparticle with abundant

hydroxyl groups could interact with QCs via electronic interactions and hydrogen

bonding, acting as an excellent sustainable drug release device at wound site.

Herein, we successfully prepared a porous sponge consisted of EW, MMT and

QCs, named as EMQS. This composite sponge displayed suitable mechanical

properties, WVTR, water uptake ability and antibacterial activity. The in vitro cell

viability evaluated by CCK8 demonstrated that EMQS was biocompatible and normal

5
human dermal fibroblast (NHDF) cells could adhere, proliferate and migrate on

EMQS with an extended morphology. To further evaluate the promotive effect for

wound healing of EMQS in vivo, three full-thickness skin defect models of BALB/c

mice were adopted, including uninfected, E.Coli and MRSA infected wounds. Wound

closure rates of the EMQS groups were significantly higher than the two

commercially available wound dressings, Tegaderm™ (for uninfected wounds) and

Atrauman AG (for infected wounds). The H&E, Masson’s and CD-31

immunohistochemical staining revealed that the EMQS treatment accelerated the

epithelialization, collagen deposition and vascularization of wounds. Moreover, the

EMQS could reduce the inflammatory response of infected wounds. The pathogen

culture of wound secretion indicated that EMQS treatment could effectively inhibit

the growth of E.Coli and MRSA at wound sites. Therefore, this composite sponge

EMQS showed the potential as a multi-functional clinical wound dressing for skin

regeneration, particularly for infected and hemorrhagic wounds.

2. Experimental Part

2.1. Materials and Methods

Chitin powder was purchased from Golden-Shell Biochemical Co. Ltd. (Zhejiang,

China). The chitin powder was purified as previously recorded [35] before use.

Potassium hydroxide (KOH) and hydrochloric acid (HCl) were purchased from

Sinopharm Chemical Reagent Co., Ltd., China. Montmorillonite was purchased from

and 2,3-epoxypropyltrimethylammonium chloride (EPTMAC) was purchased from

6
Sigma-Aldrich (USA). All reagents were of analytical grade. CCK-8 cell counting kit,

fetal bovine serum (FBS), DMEM mediums (Hyclon) were from Shanghai Yisheng

Biotechnology Co., Ltd., China.

2.2. Synthesis of quaternized chitin derivatives (QCs)

The synthesis of QCs was based on the one-pot method as previously recorded [18].

Firstly, 5g purified chitin powder was dispersed in 95g 23wt% KOH solution to obtain

a suspension and frozen at -35°C for 3 hours and thawed at room temperature

afterwards. The freezing/thawing procedure was repeated for three times to acquire a

transparent and homogenous solution, which was centrifuged at 4°C at a speed of

8000 rpm for the removal of impurities. 400g 23wt% KOH at 5°C was mixed with

chitin solution to dilute it to 1 wt%. Then, different amount of EPTMAC was added to

chitin solution (Table 1) and the resulting mixture was stirred at 10 °C for 24 h. Next,

it was neutralized with 6 mol/L HCl, and the neutral solution was dialyzed against

water using a dialysis cassette (molecular weight cut-off 8000-14000). After the

removal of salts, the QCs solution was filtered and concentrated via vacuum

distillation and finally freeze-dried.

2.3 The preparation of EW/QC/MMT composite sponges (EMQS).

The egg white protein was carefully separated from yolk and filtered with a

100-μm pore-size filter to remove impurities. Then the EW was vigorously stirred at a

speed of 1300 rad/min to generate fluffy and porous foam. MMT was dispersed into

7
deionized water at a concentration of 10 wt % and stirred and ultra-sonicated for 2

hours. And certain amount of MMT was added to EW foam while stirring. Then 2wt%

QC-3 solution was blended with foam. EMQS-1, EMQS-2 and EMQS-3 were

prepared at different weight ratios of QC-3 and MMT, which were 1:2, 1:4, 1:6

correspondingly. EMQA was prepared with EW without mechanical foaming, 2wt%

QC-3 and MMT. After 10 min of mechanical stirring and blending, the mixtures were

poured into a cylindrical mold and frozen at -40 °C for 4 h, and freeze-dried

afterwards.

2.4 Characterization

FTIR of the samples was recorded on a Perkin-Elmer FT-IR spectrometer (model

1600, PerkinElmer Co. USA) in the range of 4000-400 cm-1. The test specimens were

prepared by the KBr-disk method. The X-ray diffraction (XRD) patterns of

freezing-dried samples were recorded on an XRD instrument (XRD6000, Shimadzu,

。 。
Japan) with Cu-Kα radiation (λ= 0.154 nm). The data were collected from 5 to 80 at a

。
scanning rate of 2 /min. X-ray photoelectron spectroscopy (XPS) spectra were

recorded with an X-ray photoelectron spectrometer (ESCALAB 250Xi, Thermo

Fisher Scientific, USA). The compressive modulus of sponges were measured on

Instron MT-35.The ζ-potential of the samples in distilled water was measured on a

Nano-ZS ZEN3600 instrument (Malvern, UK) at 25 ◦C. TG profile was recorded on

TGA-Q500 (TA Instrument, USA).

The porosity of the sponges was evaluated by a liquid displacement method [5, 36].

8
Sponges were cut into regular cylindroids with a diameter of 2 cm and a height of 0.5

cm. And sponges were weighed and then immersed in absolute ethanol for 1 hours at

room temperature to allow the ethanol to permeate through the pores of the sample.

Next, the sponge was taken out from the absolute ethanol and weighed after being

wiped out the ethanol on the surface. Each sample was tested in triplicate. The

porosities of the sponges were calculated as following:

Porosity (%) = (m1 - m2)/Vρ * 100%

where m1 and m2 represent the weight of the sample before and after immersion in

ethanol, respectively. V and ρ represent the sample volume and the density of ethanol

(0.785 g/cm3).

The water uptake ability of the sponges was determined by the gravimetric method

[20]. The EMQS samples were cut into cylinders with a diameter of 2 cm and a height

of 0.5 cm. The dry sponges were weighted and then soaked in water at 37 °C for 20

min. The wet sponges were removed from water and weighted after the sponge

surface was wiped with a filter paper to remove excess water. The water uptake ability

was calculated as follows:

Water uptake ability (g/g) = (WW-Wd)/ Wd

where Wd represented the weight of dry sponges and WW represented the weight of

wet sponges.

The water vapor transmission rate (WVTR) of the sponges was determined

according to ASTM E96-00 [11]. The sponges were applied to seal the mouth of glass

bottles (10 mm diameter) containing a fixed volume of deionized water. The mass of

9
the initial state of the system was recorded as M0. The systems were placed in a

desiccator with a saturated ammonium sulfate solution at 37 °C. A glass bottle without

any covering was used as a blank control. The systems were weighed after specific

time and recorded as Mt. The WVTR was calculated as follows:

WVTR (g/m2) = (Mt- M0)/A*100

where A was the area of the glass bottle mouth (m2).

The release efficiency of QC-3 incorporated with MMT was measured. 2 wt%

QC-3 aqueous solution was blended with 10 wt% MMT solution at the weight ratio of

1:4. The samples were freeze-dried and weighed (marked as M0). Then samples were

immersed into PBS solution for different times. After immersion, the sample was

centrifuged and the liquid supernatant was carefully removed. The remained sample

was washed with deionized water and centrifuged. Finally, the remained sample was

freeze-dried and weighed, marked as Mt. The release efficiency was calculated as

follow:

Release efficiency (%) = (M0 - Mt)/ (M0 * 0.2) *100

The in vitro degradation was measured. Briefly, the sponge samples were immersed

in 100 mg/mL lysozyme solutions at 37 °C with shaking at 100 rpm. After

predetermined time intervals, the samples were taken out and washed three times.

Then the samples were lyophilized and weighed. The weight remaining at particular

time point was calculated as follows:

Weight remaining (%) = m2/m1 * 100%

where m1 and m2 represent the weight of the initial and final sponges, respectively.

10
2.5 Cytotoxicity and Cell Culture

Normal human skin fibroblasts (NHDF) was used to conduct cytocompatibility

assay. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)

containing 1% (v/v) of penicillin (100units/mL) and streptomycin (100 units/mL)

solution and 10% (v/v) fetal bovine serum (FBS) in an incubator at 37°C with 5%

CO2. Sponges were cut into a regular shape and autoclaving sterilized before being

placed in the wells of 96-well plates, with some wells left empty as control groups.

NHDF cells were seeded in 96-well plates at a density of 1*104 cells/wells, and 200

μL of cell suspension was added to each well for co-culture with sponges. After

culturing for different periods of time, 20μL of CCK-8 was added into each well

followed by incubation for 15 min. 100 μL solution of each well was transferred into a

new plate accordingly. The absorbance value A at the wavelength of 488 nm was

detected by ELIASA. The cell viability was calculated as following equation：

Cell viability (%) = Atest / Acontrol × 100%

Where the Atest and Acontrol represent the absorbance values of groups co-cultured with

sponges and the control groups cultured alone.

2.6. In vitro antimicrobial test

The in vitro antimicrobial properties of the materials were quantitatively evaluated

with bacterial plate coating. MRSA and E. coli were cultured in a standard

Luria-Bertani (LB) culture medium [37]. Freeze-dried EW, MMT and EMQS-2 at a

11
weight ratio of 1 g/mL were added into 1 mL MRSA and E. coli solutions of 5 × 106

CFU/mL, respectively. Microbial suspensions without QCs were used as negative

controls.

2.7 In vivo wound healing full-thickness skin defect and MRSA, E.Coli infected

wounds model

The wound-healing efficacy of the samples was evaluated in male BALB/C mice

(weighing 20 ± 2 g) supplied by the Animal Experimental Center of Tongji Medical

College, Huazhong University of Science and Technology. All procedures were

carried out in compliance with the guide for the care and use of laboratory animal

resources and the national research council, and approved by the Institutional Animal

Care and Use Committee at Tongji Medical College, Huazhong University of Science

and Technology (2423). All mice were housed in a controlled environment (12 hours

light/dark cycle at 21°C) with free access to water and standard chow diet. After 7

days of adjusting to the new environment, the back of the mice were shaved and use

70% alcohol and iodophor disinfectant solutions to disinfect the dorsal skin of mice.

After anesthesia, a 1.0 × 1.0 cm2 full-thickness skin and subcutaneous panniculus

carnosus was removed. A total of 108 full-thickness skin defect mice were used to

complete three parts of the animal experiment. Part 1, noninfectious wound

experiment, including three groups, wounds were covered with gauzes, commercial

dressings (Tegaderm™, 3M, USA) and EMQS-2. Part 2, methicillin-resistant S.

aureus (MRSA) infected wounds, the wounds were inoculated with 0.1ml 108 CFU/ml

12
MRSA (ATCC43300) after the full-thickness skin defect, and were covered with

gauzes, commercial antibacterial dressings (Atrauman AG, PAULHARTMANNAG,

Germany) and EMQS-2 at three days later. Part 3, Escherichia coli (E.Coli) infected

wounds, the wounds were inoculated with 0.1ml 108 CFU/ml E.Coli (ATCC25922)

after the full-thickness skin defect, and were covered with gauzes, commercial

antibacterial dressings (Atrauman AG, PAULHARTMANNAG, Germany) and

EMQS-2 at three days later. Each group contained twelve mice.

2.8 Wound Closure Analysis

Macrophotography of the mice’s wounds and a metric ruler were photographed on

days 0, 3, 5, 7, 9, 11 and 14. Time to wound closure was defined as the time taken for

the wound bed to be completely reepithelialized. Wound area was measured and

calculated as described previously using digital planimetry (Image J; National

Institutes of Health, Bethesda, Md.). 3 skin samples including the wound and 5mm

margin of surrounding skin were harvested at 0, 3, 7, and 14 days after wounding and

were bisected. Half was processed for histologic and immunohistologic studies and

the other half was snap-frozen in liquid nitrogen for further western-Blot studies.

2.9 Histology and Morphologic Studies

For the evaluation of epidermal regeneration in wound area and collagen deposition,

tissues were processed as described previously. Representative sections were stained

with Hematoxylin and eosin (H&E), Masson’s trichrome (Masson). All slices were

13
taken and analyzed using a digital microscope system (Aperio VERSA 8, LEICA).

The proportion of collagen-occupied area (%/5×field) was measured on histological

skin samples by Image J software.

2.10 Immunohistochemistry for CD-31

Paraffin-embedded sections were rehydrated and antigen retrieval for CD-31 were

performed by microwaving in EDTA buffer. Primary antibodies used were as follows:

CD-31(Affinity Biosciences, USA). Secondary antibody used were horseradish

peroxidase–conjugated goat anti-mice immunoglobulin G (Aspen, USA). Primary

antibodies were incubated at 4°C overnight. Sections were counterstained with

haematoxylin.

2.11 Blood Vessel Density Quantitation

To quantify angiogenesis, twelve fields, including bilateral leading edges (six fields)

as well as the middle part of each CD-31-stained wound, were evaluated at ×100

magnification. The neovascular area (CD-31) were measured using Image J and

expressed as the percentage of CD-31 area of the entire imaged area. Measurements

were obtained using Image J software by two independent and blinded observers.

2.12 Bacterial culture of wound secretion

The secretions on the wound of the MRSA and E.Coli infection mice were collected

for examination, and microbial culture was conducted. Pathogen identification and

14
isolation were conducted with automatic microbial assay instrument, and the detection

rate of pathogenic bacteria was calculated.

2.13 White blood cell count of mice

Blood was collected from the tail vein of each mouse into the EDTA vacuum tubes.

Complete blood count (CBC) including RBC count, hemoglobin (Hb), hematocrit

(Hct), mean corpuscular volume (MCV), mean cell hemoglobin (MCH), mean cell

hemoglobin concentration (MCHC), RBC distribution width (RDW), reticulocytes,

white blood cell (WBC) count, and WBC differential was subsequently performed

using the automatic hematology analyzer(BC-2800vet, Mindray, CHINA).

2.14 Western blotting

Fifty milligrams of wound tissue was homogenized in ice-cold buffer containing 1%

Triton X-100, 100 mM NaCl, 20 mM Tris pH 7.5, 2.0 mM EDTA, 10 mM MgCl2, 10

mM NaF, 40 mM b-glycerol phosphate, 1 mM PMSF, 2 mM sodium orthovanadate,

protease inhibitor cocktail tablets (Roche Diagnostics, Indianapolis, IN), 10 mg/mL

leupeptin, and 7 mg/mL pepstatin. The homogenates were treated with 1% SDS

(Triton-X insoluble fraction), boiled and centrifuged to collect the supernatant.

Protein concentrations were determined using a BCA protein quantitation kit (Thermo

Scientific, Rockford, IL). Add 10 times the volume of tissue protein extraction reagent,

homogenize thoroughly in ice bath, and collect the total protein solution. After

SDS-PAGE electrophoresis, the proteins were transferred onto a PVDF membrane

15
(0.45um) (Millipore, USA), blocked with 5% nonfat milk in TBS-T buffer for 2 h at

37 ℃, and incubated overnight at 4 ℃ with primary anti-bodies against the target

proteins. Primary antibodies used were as follows: β-Actin (TDR BIOTECH,

CHINA), IL-1 (abcam, USA), IL-6 (Affinity, USA), IL-10 (proteintech, USA), TNF-α

(Zen Bioscience, CHINA)， TGF-β (Huabio, CHINA), VEGF (Zen Bioscience,

CHINA). The membrane was then incubated with the appropriate HRP-conjugated

secondary antibody (ASPEN, USA) and developed with the ECL system (ASPEN,

USA). All experiments were repeated 3 times, and one representative blot is shown.

2.15 The hemostatic effect of particles in vivo

Male SD rats (2-3 months, weight ≈ 250 g) were anesthetized by intraperitoneal

injection (0.4 mL/100 g) of 10% chloral hydrate. Hair was removed from the liver of

SD rats and the surgical area was disinfected with 75% alcohol. The liver was cut 0.5

cm with a scalpel to make the liver injury model. The wound was immediately

covered with the sample (200 mg) after the blood was wiped clean with sterile gauze,

and the bleeding time and blood loss weight were recorded. The blank group absorbed

blood with sterile gauze, and repeated 6 times for each sample. All procedures were

carried out in compliance with the guide for the care and use of laboratory animal

resources and the national research council, and approved by the Animal Biosafety

Level 3 Laboratory of Wuhan University Institutional Animal Care and Use

Committee.

16
2.16 Statistical Analysis

All statistical data are presented as mean ± SD. Data comparisons were performed

by t tests and two-way analysis of variance using SPSS Version 17.0 (SPSS, Inc.,

Chicago, Ill.). A value of p < 0.05 was considered significant.

3. Results and Discussion

3.1 The foaming of egg white protein (EW) and the characterization of composite

dressing

A clinical tissue engineering scaffold should combine the ideal biochemical and

biophysical properties, mechanical adaptability and surface morphology, promoting

favorable cellular interactions and tissue development. In this study, egg white protein

(EW) was applied as a foaming agent to create a porous morphology of the sponge

because the appropriate porosity of the wound dressing benefit exudates adsorption,

gas exchange, water evaporation and cell proliferation and migration [20, 30]. Fig. 1(a)

showed the roadmap of EW foam transformation from the liquid state by mechanical

stirring, followed by the incorporation of montmorillonite (MMT) and the quaternized

chitin derivatives (QCs). And finally the composite sponge, EMQS, was prepared via

the freeze-drying method. As the SEM images (Fig. 1(b)) showed, the non-foaming,

directly freeze-fried composite aerogel (EMQA) exhibited a denser surface and

smaller pore size of about 50-100 μm, lack of an inter-connected structure, while

EMQS-1 had a well-developed pore network structure, showing numerous open

interconnected cells with a wider pore size distribution (50-300 μm). And the resultant

17
increasing porosity (from 55.3% to 84.8%) led to better water uptake ability (from

521.4 to 675.3 g/g) and water vapor transmission rate (WVTR) at the same time point.

The water contact angle of EMQA and EMQS-1 (Fig. 1(c)) demonstrated that the

hydrophobicity of the dressing was enhanced (from 43.3°to 71.2°). This might be

attributed to the conformational change of proteins during mechanical stirring. The

hydrophobic domains of proteins were exposed and proteins aggregated at the

air-water interface, as a colloidal system containing tiny air bubbles dispersed in an

aqueous continuous phase [38, 39]. The FTIR spectrum of freeze-dried EW and EW

foam (Fig. 1(d)) were applied to examine the chemical and physical changes, as the

bands of amide I and II are able to assess the alterations in the secondary structures of

proteins [40]. The peaks at 1648 cm-1 of amide I of EW, 1532 cm-1 of amide II [41]

and the band at 3303 cm-1 assigned to hydrogen bonded N-H stretching didn’t shift

after foaming process, indicating no drastic damage of the intermolecular hydrogen

bonds of EW. The foaming process was a physical transition without changing the

components with beneficial effect of EW for promoting wound healing.

The foaming process, creating higher porosity and larger pore size, adversely

effected the mechanical properties of composite sponges, and the compressive

strengths decreased from 0.155 (EMQA) to 0.056 MPa (EMQS-1) (Fig. 1(e)). Hence,

we tried to increase the amount of MMT, as a reinforcement nano-building block, to

enhance the mechanical properties. With the increase of MMT (Table S1), sponges

showed enhanced compressive strengths, 0.067 MPa of EMQS-2 and 0.126 MPa of

EMQS-3. On the other hand, the porosity of sponges was adversely influenced with

18
more MMT added (Fig. 1(f)). The porosity of EMQS-2 and EMQS-3 decreased to

78.9% and 73.2% respectively (Table S2). And water uptake ability (Fig. 1(g)) and

WVTR (Fig. 1(h)) showed the same declining trend, as porosity is the main factor that

impacts the adsorption and water transmission ability of sponge materials [20].

The interaction between MMT and QC-3 was studied via XPS, FTIR, TG and XRD.

We utilized QC-3 as the material for the fabrication of composite dressing considering

that it had the highest degree of quaternization (DQ) (0.48) and positive ζ-potential

(59.57 mV) (Table S1). And this would be beneficial for the interaction with the

negatively charged MMT, together as a sustainable drug release system. Due to the

abundant anionic charges on the surface of MMT, this medical clay has been adopted

to interact with many cationic drugs or polymers via multiple interactions including

electrostatic interactions, hydrogen bonding and van der waals, which was used for

controlled drug release such as antibiotics, anticancer drugs and even bioactive

proteins [42, 43]. The full X-ray photoelectron spectroscopy (XPS) spectra in Fig. 2(a)

showed the overall atomic content of C, N, and O in different samples. The oxygen in

QC-3 was distributed into three typical forms, C-O- (530.8 eV), -O-H (531.9 eV), and

C=O (533.2 eV) [44]. As Fig. 2(b) and (c) showed, the shift of the C=O peak from

533.2 eV to 532.7 eV confirmed the formation of the hydrogen bonding between

QC-3 and MMT [45]. The peak at 402.4 eV for nitrogen in -N+ (CH3)3 showed a

minor shift of 0.2 eV (402.6 eV) (Fig. 2(d)), indicating that quaternary ammonium

groups were scarcely involved in the hydrogen bonding interplay, probably due to the

steric hindrance. FTIR spectrum of MMT at 3620 cm-1 assigned to the stretch of -OH

19
in Al-OH (Fig. 2(e)), was restrained after the incorporation of QC-3, indicating the

formation of hydrogen bonds via the hydroxyl groups of MMT. The peak of 1640

cm-1 that is attributed to -OH bending mode of adsorbed water [46] was overlapped

with the amide of QC-3 at 1650 cm-1. The characteristic peaks at 1115 and 1035 cm-1

were assigned to Si-O stretching (out-of-plane) and Si-O stretching (in-plane)

vibration for MMT [47]. After mixing with QC-3, the 1115 cm-1 peak shifted to 1105

cm-1 while 1035 cm-1 peak remained its original position. This result indicated that the

binding of QC-3 via hydrogen bonding occurred outside the plane of MMT and QC-3

was not intercalated into MMT. The peaks at 1560 cm−1 and 1316 cm−1 corresponded

to the amide II and III of QC-3 [19] shifted to lower wave number (1550 and 1310

cm−1) as an evidence of the intermolecular hydrogen bonding and electrostatic force

between QC-3 and MMT. The characteristic 2θ peak of (001) plane for MMT was

observed at 7.4° [48]. After the incorporation of QC-3, the (001) plane disappeared.

Hence, unlike the general method of cationic drugs or polymers intercalated into

MMT layers via cationic exchange mechanisms that generally caused the increase of

interplanar distance [49], QC-3 seemed to be simply physically cross-linked by MMT

via hydrogen bonding and electrostatic interactions and MMT aggregated into

disordered structure, possibly due to the insufficient mixing time and high molecular

weight of QC-3 [29]. TG profile (Fig. 2(g)) of QC-3 showed the initial weight-loss

temperature at 219.5 ℃, while the initial weight-loss temperature of MMT/QC-3

increased to 240.5 ℃, indicating the formation of MMT-QC-3 network. The QC-3

and MMT formed the polymer-nano inorganic particles network through the

20
electrostatic interactions between the protonated amino groups of the QC-3 and the

anionic charged groups at surface of the MMT, and hydrogen bonding generated

through the abundant hydroxyl groups of QC-3 and MMT.

As a consequence, the polymer network physically cross-linked by MMT was able

to release gradually in aqueous solution instead of dissolving immediately. And it was

proved that the release of antibacterial agents in a controlled manner could more

effectively inhibit the growth of bacteria [50]. Fig. 2(h) displayed the release

efficiency of QC-3/MMT in PBS solution. By measuring the remaining weight of

samples after different period of immersion time, it was clearly that QC-3 was slowly

released into the ambient. About 81.8% of QC-3 was released from the freeze-dried

QC-3/MMT samples after 36 h. In comparison, the dissolution time of pure QC-3 at a

concentration of 1 wt% was within half an hour. Therefore, by the formation of the

physical-crosslinked polymer net-work, QC-3 was able to be released in a controlled

manner. This characteristic make the EMQS a clinical dressing candidate. The sponge

dressing could quickly absorb the blood and exudates at wound site, and then QC-3

could be gradually dissolved for a constant period of time. MMT provided great

mechanical properties and maintained the integrity of dressing until it needed

changing.

3.2 Cytocompatibility, antibacterial ability and hemostatic effect of composite

dressing

Considering the suitable mechanical properties, water uptake ability and WVTR

21
in general, we chose EMSQ-2 as the dressing for cytocompatibility assessment and

wound treatment in vivo. The primary requirement for a wound dressing is

biocompatibility. A qualified wound dressing should be capable of offering an

environment where cells can attach, proliferate and migrate for the regeneration of

damaged tissue. Normal human skin fibroblasts (NHDF) were adopted to assess the

cytocompatibility of EMQS. The CCK8 evaluation confirmed that EMQS with

different amount of MMT was non-toxic to NHDF, of which the viabilities reached to

over 95% after co-culture with EMQS at the time point of 24, 72 and 120h (Fig. 3(a)).

The SEM images of NHDF cultivated on EMQS-2 for 48h (Fig. 3(b-d)) indicated that

this composite sponge was capable to support cell adhesion, proliferation and

migration. NHDF exhibited an extended morphology on EMQS with a high cell

proliferation ratio. And NHDF migrated expansively into the sponge owing to the

interconnected porous architecture that offered an accessible ambient. This migration

of fibroblasts is crucial for the formation of granulation tissue and the deposition of

collagen at wound sites [51].

The antibacterial activities of EW, MMT, QCs and EMQS-2 were evaluated via the

spread plate method (Fig. 4). As the results, the minimal inhibition concentration

(MIC) of QCs was related to the degree of quaternization (Fig. S3), consistent with

the previous records. QC-3 had the lowest MIC, 5 μg/mL against E.Coli and 10

μg/mL against MRSA after 2h sterilization. EW also showed antibacterial ability,

about 71.4% against E.Coli and 74.3% against MRSA at a concentration of 1 g/mL,

indicating that this freeze-drying method didn’t sabotage the activity of the main

22
antibacterial substances in EW. The inconspicuous antibacterial activity of MMT was

probably attributed to the absorption effect to bacteria via electronical interactions

[52]. And the EMQS-2 displayed a superior antibacterial ability of 97.6% to E.Coli

and 98.4% to MRSA.

The liver injury models of Sprague-Dawley (SD) rats were established in order to

further investigate the hemostatic effect of EMQS-2. The blood loss weight were

determined to explain the hemostasis ability of EMQS-2 and the results were shown

in Fig. 5. It was found that EMQS-2 treatment could significantly reduce the blood

loss weight and shorten the bleeding time. Compared with the untreated group (163.0

± 18.9 mg), EMQS-2 reduced the blood loss to 52.7 ± 9.1 mg (P < 0.01), and

shortened the hemostasis time from102.5 ± 13.7 s (untreated group) to 49.7 ± 8.0 s (P

< 0.01).

3.3 Wound healing capacity of EMQS-2

The ability of EMQS-2 to promote wound healing was studied in a full-thickness

skin defect model, a MRSA infected and an E.Coli infected full-thickness skin defect

model. Each model had three groups as parallel test, which included a control group

treated with gauze, an experimental group treated with EMQS-2 and a commercial

dressing treated group (Tegaderm™ for uninfected wound and Atrauman AG for

infected wounds). And the quantitative calculation of wound area at different healing

time was conducted to accurately depict the promotive effect of EMQS-2 compared to

other dressings.

23
 As shown in Fig. 6 (a1)-(a3), the macroscopic observations of wounds indicated

that the wound area of all groups started to decrease significantly from day 3. The

area of uninfected wounds treated with EMQS-2 reduced almost at the same rate as

that treated with Tegaderm™ (P>0.05) (Fig. 6 (b1)). And wounds treated with

EMQS-2 displayed a much more rapid closure rate than those treated with gauze on

the day 11 and 14 (P<0.01)). The MRSA infected wounds treated with EMQS-2 group

began to show an obviously faster wound area reduction than other groups on day 3,

and this trend continued, with a statistical difference compared to the gauze group and

Atrauman AG group, until day 14 (p<0.01) (Fig. 6(b2)). As for the E.Coli infected

wound model, the EMQS-2 group began to show a rapid reduction in open wound

area from day 3 on, faster than other two groups. And the wound size measurements

of EMQS group had a statistical difference compared to the gauze group and

Tegaderm™ group on the day 7, 9, 11, 14 (p<0.01) (Fig. 6 (b3)).

3.4 Immunohistochemical staining analysis

H&E staining and Masson’s trichrome staining were used for histomorphological

examination of epithelialization and collagen deposition during the wound healing

process (Fig. 7). H&E staining results (Fig. 7(a)) showed that the EMQS-2 groups had

a significantly faster rate of skin regeneration than those of gauze groups and the

Tegaderm™/Atrauman AG groups (the length of black lines represented the unhealed

area) in all models. In addition, the Masson’s trichrome staining profiles showed that

more newly produced collagen, the purple staining in Fig. 7(b1), was deposited in the

24
wounds treated with EMQS-2 than in the wounds treated with gauze, Tegaderm™ and

Atrauman AG (p<0.05) (Fig. 7(b2)).

The evaluation of angiogenesis was conducted to show the accelerated healing

effects of EMQS-2 via CD-31+ staining (Fig. 8(a)), and angiogenesis at the 14th day

was determined by quantification of blood vessel density (Fig. 8(b)). Rate of CD-31+

areas of uninfected wound edge showed increased in EMQS-2 groups compared with

the Tegaderm™ group in the uninfected wounds on day 14 (p<0.05) (Fig. 8(b1)). And

rates of CD-31+ areas of wounds edge in EMQS-2 groups of E.Coli-infected and

MRSA-infected wounds on day 14 were higher compared with the gauze groups and

the Atrauman AG groups (p<0.01) (Fig. 8(b2) and (b3)).

Among the variety of factors, VEGF and TGF-β are the major growth factors

involved in inflammatory and proliferative phase, able to promote epithelization,

angiogenesis, cell migration and differentiation [53]. The levels of growth factor

VEGF and TGF-β in the wound edges of each groups were detected by Western Blot

on the 7th day to study the mechanism of enhanced angiogenesis after EMQS-2

treatment. One representative blot of VEGF and TGF-β were shown in Fig. 9(a). In

the uninfected wounds, the protein expression of VEGF in the EMQS-2 group were

higher than those both in the gauze group and the Tegaderm™ group (p < 0.05) and

the expression of TGF-β in the EMQS-2 group was higher than that in the gauze

group (p < 0.001) (Fig.9(a1)). In the E.Coli-infected and MRSA-infected wounds, the

expression of VEGF and TGF-β in the EMQS-2 group were higher than both gauze

groups and the Atrauman AG groups (p < 0.05). The higher level of TGF-β likely

25
improved the proliferation of fibroblasts, which produced more collagen and

increased collagen density at the wound site [54].

3.5 The capacity of EMQS-2 to alleviate inflammatory response

To understand the possible underlying mechanism of the promotive effect of

EMQS-2 in wound healing, we conducted a series of tests at different wound healing

stage. As we all know, wound healing is a dynamic and complex process of tissue

regeneration and growth progress involving interactions of cytokines, growth factors,

blood, multiple components in extracellular matrix [55]. Generally speaking, it could

be divided into four overlapping but well-defined phases: hemostasis, inflammation,

proliferation and remodeling [51]. In the inflammation phase, the bacterial infection

of a persistent tissue level should be rigorously avoided. It would cause the production

of endotoxin and result in the prolonged elevation of proinflammatory cytokines [56],

and the self-perpetuating inflammatory cascade may increase tissue destruction and

necrosis rather than healing [57]. Wound secretions were collected for pathogen

culture in the E.Coli-infected and MRSA-infected wounds on the 3rd, 7th, 14th day to

evaluate the anti-bacterial ability of the EMQS-2 in vivo. In EMQS-2 group, with the

extension of time, the detection rate of E.Coli and MRSA dropped sharply. Until the

14th day, the detection rate of bacteria was zero (Fig. 10(a) and (b)), indicating the

complete elimination of pathogen at wound sites. In comparison, the gauze group and

the Atrauman AG groups showed a much higher detection rate of pathogenic bacteria.

The alteration of hematological parameters of White Blood Cells (WBC) in

26
E.Coli-infected and MRSA-infected mice treated with EMQS-2 were presented in Fig.

10(c) and (d). As a serum inflammatory marker, WBC can reflect the systemic

inflammatory response in animals to a certain extent (limited diagnostic value of

serum inflammatory biomarkers in the diagnosis of fracture-related infections).The

decrease of WBC was observed in EMQS-2 groups compared with the gauze and

Atrauman AG groups on the day 3,7,14 (p < 0.05). This implied that EMQS restored

WBC homeostasis in mice infected with E.Coli and MRSA.

Chronic inflammation is a negative process for tissue regeneration, which would

delay the wound healing process. The expressions of inflammation related biomarkers

in the wounds clarified the mechanism of the lightened inflammatory reaction (Fig.

9(a)). IL-1, IL-6, IL-10 and TNF-α are the main inflammatory mediators in the phase,

of which the expression level indicated the time of phase duration. IL-1, IL-6 and

TNF-α have been proved to hinder the diabetic wound healing by increasing

macrophage and neutrophil infiltration [58, 59], while IL-10, an anti-inflammatory

factor, was in favor of tissue repair and regeneration [60]. In the present study, the

expression of IL-1, IL-6 and TNF-α in EMQS-2 group were lower than gauze group

both in E.Coli-infected and MRSA-infected wounds at the 7th day (p < 0.05). In

E.Coli-infected wounds, there was no difference on the expression of IL-1, IL-6 and

IL-10 in EMQS-2 group compared with those in Atrauman AG group (p > 0.05). But

the expression of TNF-α in EMQS-2 group were lower than Atrauman AG group.

While in MRSA-infected wounds, the expression of IL-6 and TNF-α in EMQS group

were lower than Atrauman AG group (p < 0.001). On the contrary, the expression

27
level of IL-10 in EMQS-2 group was higher than that in Atrauman AG group (p <

0.001). This could conclude that the EMQS-2 treatment effectively inhibited the

growth of pathogens and decreased the expression of some inflammatory factors, and

thus shorten the inflammatory phase.

Conclusion

In this research, EMQS could serve as a multi-functional dressing and promote the

wound healing of infected wounds. It exhibited porous structure with a porosity of

84.8%, good mechanical performance (0.126 MPa of compressive strength) and water

uptake ability (675.3 g/g). The excellent hemostatic ability (hemostatic time of 49.7 ±

8.0 s in a liver injury model) could rapidly reduce the blood loss of acute wounds.

This natural protein and polysaccharide based dressing could prohibit the growth of

pathogen at wound sites via the sustainable release of QC, and the inflammation

reaction was alleviated after the application of EMQS in the infected wounds. The

enhanced expression of growth factors including VEGF and TGF-β after EMQS

treatment led to faster re-epithelization, stronger angiogenesis and collagen deposition

at dermal layer. As a result, the EMQS could promote the hemorrhagic and infected

wound healing. Hence, this all-natural substances based, cheap and bio-safe wound

dressing had a huge potential to be adopted clinically.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of

28
China (21620102004 ， 22075214, 52003204) ， Key Research and Development

Program of Hubei Province China (2020BCA079), Fundamental Research Funds for

the Central Universities (2042020kf0040) and the Health Commission of Hubei

Province Medical Leading Talent Project (Grant No. LJ20200405)

References

[1] Simões D, Miguel SP, Ribeiro MP, Coutinho P, Mendonça AG, Correia IJ. Recent
advances on antimicrobial wound dressing: A review. European Journal of
Pharmaceutics and Biopharmaceutics. 2018;127:130-41.
[2] Chua AWC, Khoo YC, Tan BK, Tan KC, Foo CL, Chong SJ. Skin tissue
engineering advances in severe burns: review and therapeutic applications. Burns &
Trauma. 2016;4(1):3.
[3] Rezvani Ghomi E, Khalili S, Nouri Khorasani S, Esmaeely Neisiany R,
Ramakrishna S. Wound dressings: Current advances and future directions. Journal of
Applied Polymer Science. 2019;136(27):47738.
[4] Ye S, Jiang L, Wu J, Su C, Huang C, Liu X, et al. Flexible Amoxicillin-Grafted
Bacterial Cellulose Sponges for Wound Dressing: In Vitro and in Vivo Evaluation.
ACS Appl Mater Interfaces. 2018;10(6):5862-70.
[5] Che C, Liu L, Wang X, Zhang X, Luan S, Yin J, et al. Surface-Adaptive and
On-Demand Antibacterial Sponge for Synergistic Rapid Hemostasis and Wound
Disinfection. ACS Biomaterials Science & Engineering. 2020;6(3):1776-86.
[6] Yang H, Liang Y, Wang J, Li Q, Li Q, Tang A, et al. Multifunctional wound
dressing for rapid hemostasis, bacterial infection monitoring and photodynamic
antibacterial therapy. Acta Biomaterialia. 2021;135:179-90.
[7] Zhao X, Wu H, Guo B, Dong R, Qiu Y, Ma PX. Antibacterial anti-oxidant
electroactive injectable hydrogel as self-healing wound dressing with hemostasis and
adhesiveness for cutaneous wound healing. Biomaterials. 2017;122:34-47.
[8] Liu L, Shi J, Sun X, Zhang Y, Qin J, Peng S, et al. Thermo-responsive
29
hydrogel-supported antibacterial material with persistent photocatalytic activity for
continuous sterilization and wound healing. Composites Part B: Engineering.
2022;229:109459.
[9] Zhao N, Yuan W. Highly adhesive and dual-crosslinking hydrogel via one-pot
self-initiated polymerization for efficient antibacterial, antifouling and full-thickness
wound healing. Composites Part B: Engineering. 2022;230:109525.
[10] Li Y, Wang J, Wang Y, Cui W. Advanced electrospun hydrogel fibers for wound
healing. Composites Part B: Engineering. 2021;223:109101.
[11] Ma R, Wang Y, Qi H, Shi C, Wei G, Xiao L, et al. Nanocomposite sponges of
sodium alginate/graphene oxide/polyvinyl alcohol as potential wound dressing: In
vitro and in vivo evaluation. Composites Part B: Engineering. 2019;167:396-405.
[12] Tchobanian A, Van Oosterwyck H, Fardim P. Polysaccharides for tissue
engineering: Current landscape and future prospects. Carbohydrate Polymers.
2019;205:601-25.
[13] Graça MFP, Miguel SP, Cabral CSD, Correia IJ. Hyaluronic acid—Based wound
dressings: A review. Carbohydrate Polymers. 2020;241:116364.
[14] Wu J, Su C, Jiang L, Ye S, Liu X, Shao W. Green and Facile Preparation of
Chitosan Sponges as Potential Wound Dressings. ACS Sustainable Chemistry &
Engineering. 2018;6(7):9145-52.
[15] Luo X, Liu Y, Pang J, Bi S, Zhou Z, Lu Z, et al. Thermo/photo dual-crosslinking
chitosan-gelatin methacrylate hydrogel with controlled shrinking property for
contraction fabrication. Carbohydrate Polymers. 2020;236:116067.
[16] Tavakolizadeh M, Pourjavadi A, Ansari M, Tebyanian H, Seyyed Tabaei SJ,
Atarod M, et al. An environmentally friendly wound dressing based on a self-healing,
extensible and compressible antibacterial hydrogel. Green Chemistry.
2021;23(3):1312-29.
[17] Wang L, Zhang X, Yang K, Fu YV, Xu T, Li S, et al. A Novel Double‐
Crosslinking‐Double‐Network Design for Injectable Hydrogels with Enhanced Tissue
Adhesion and Antibacterial Capability for Wound Treatment. Advanced Functional
Materials. 2019;30(1):1904156.
30
[18] Xu H, Fang Z, Tian W, Wang Y, Ye Q, Zhang L, et al. Green Fabrication of
Amphiphilic Quaternized β-Chitin Derivatives with Excellent Biocompatibility and
Antibacterial Activities for Wound Healing. Advanced Materials.
2018;30(29):1801100.
[19] Ding F, Shi X, Li X, Cai J, Duan B, Du Y. Homogeneous synthesis and
characterization of quaternized chitin in NaOH/urea aqueous solution. Carbohydrate
Polymers. 2012;87(1):422-6.
[20] Gao H, Zhong Z, Xia H, Hu Q, Ye Q, Wang Y, et al. Construction of cellulose
nanofibers/quaternized chitin/organic rectorite composites and their application as
wound dressing materials. Biomaterials Science. 2019;7(6):2571-81.
[21] Liu X, Niu Y, Chen KC, Chen S. Rapid hemostatic and mild polyurethane-urea
foam wound dressing for promoting wound healing. Materials Science and
Engineering: C. 2017;71:289-97.
[22] Lu T, Zou Q, Zhu K, Yuan D, Ma M, Ye C. Electrospun egg white/polyvinyl
alcohol fiber dressing to accelerate wound healing. Journal of Polymer Research.
2021;28(2):1-15.
[23] Deng C, Shao Y, Xu M, Yao Y, Wu N, Hu H, et al. Effects of metal ions on the
physico-chemical, microstructural and digestion characteristics of alkali-induced egg
white gel. Food Hydrocolloids. 2020;107:105956.
[24] Geng F, Huang X, Ma M. Hen egg white ovomacroglobulin promotes fibroblast
migration via mediating cell adhesion and cytoskeleton. Journal of the Science of
Food and Agriculture. 2016;96(9):3188-94.
[25] You R, Zhang J, Gu S, Zhou Y, Li X, Ye D, et al. Regenerated egg white/silk
fibroin composite films for biomedical applications. Materials Science and
Engineering: C. 2017;79:430-5.
[26] Jalili-Firoozinezhad S, Rajabi-Zeleti S, Mohammadi P, Gaudiello E, Bonakdar S,
Solati-Hashjin M, et al. Facile fabrication of egg white macroporous sponges for
tissue regeneration. Adv Healthc Mater. 2015;4(15):2281-90.
[27] Li Q, Lu F, Zhou G, Yu K, Lu B, Xiao Y, et al. Silver Inlaid with Gold
Nanoparticle/Chitosan Wound Dressing Enhances Antibacterial Activity and Porosity,
31
and Promotes Wound Healing. Biomacromolecules. 2017;18(11):3766-75.
[28] Shao Y, Zhao Y, Xu M, Chen Z, Wang S, Tu Y. Effects of copper ions on the
characteristics of egg white gel induced by strong alkali. Poultry Science.
2017;96(11):4116-23.
[29] Feng G, Jiang F, Hu Z, Jiang W, Liu J, Zhang Q, et al. A novel porous egg-white
(EW)/titania composite photocatalytic material for efficient photodegradation
applications. RSC Advances. 2020;10(14):8525-9.
[30] Deng P, Jin W, Liu Z, Gao M, Zhou J. Novel multifunctional adenine-modified
chitosan dressings for promoting wound healing. Carbohydr Polym.
2021;260:117767.
[31] Wang Q, Zhou S, Wang L, You R, Yan S, Zhang Q, et al. Bioactive silk fibroin
scaffold with nanoarchitecture for wound healing. Composites Part B: Engineering.
2021;224:109165.
[32] Podsiadlo P, Kaushik AK, Arruda EM, Waas AM, Shim BS, Xu J, et al.
Ultrastrong and Stiff Layered Polymer Nanocomposites. Science.
2007;318(5847):80-3.
[33] Polat TG, Duman O, Tunç S. Agar/κ-carrageenan/montmorillonite
nanocomposite hydrogels for wound dressing applications. International Journal of
Biological Macromolecules. 2020;164:4591-602.
[34] Liu C, Liu C, Yu S, Wang N, Yao W, Liu X, et al. Efficient antibacterial
dextran-montmorillonite composite sponge for rapid hemostasis with wound healing.
International Journal of Biological Macromolecules. 2020;160:1130-43.
[35] Wu T, Huang L, Sun J, Sun J, Yan Q, Duan B, et al. Multifunctional chitin-based
barrier membrane with antibacterial and osteogenic activities for the treatment of
periodontal disease. Carbohydrate Polymers. 2021;269:118276.
[36] Sathiyaseelan A, Shajahan A, Kalaichelvan PT, Kaviyarasan V. Fungal chitosan
based nanocomposites sponges—An alternative medicine for wound dressing.
International Journal of Biological Macromolecules. 2017;104:1905-15.
[37] Zhu M, Liu X, Tan L, Cui Z, Liang Y, Li Z, et al. Photo-responsive
chitosan/Ag/MoS2 for rapid bacteria-killing. Journal of Hazardous Materials.
32
2020;383:121122.
[38] Babaei J, Mohammadian M, Madadlou A. Gelatin as texture modifier and
porogen in egg white hydrogel. Food Chemistry. 2019;270:189-95.
[39] Damodaran S. Protein Stabilization of Emulsions and Foams. Journal of Food
Science. 2010;70(3).
[40] Babaei J, Khodaiyan F, Mohammadian M. Effects of enriching with gellan gum
on the structural, functional, and degradation properties of egg white heat-induced
hydrogels. International Journal of Biological Macromolecules. 2019;128:94-100.
[41] Nasabi M, Labbafi M, Mousavi ME, Madadlou A. Effect of salts and nonionic
surfactants on thermal characteristics of egg white proteins. International Journal of
Biological Macromolecules. 2017;102:970-6.
[42] Jayrajsinh S, Shankar G, Agrawal YK, Bakre L. Montmorillonite nanoclay as a
multifaceted drug-delivery carrier: A review. Journal of Drug Delivery Science and
Technology. 2017;39:200-9.
[43] Kamari Y, Ghiaci P, Ghiaci M. Study on montmorillonite/insulin/TiO2 hybrid
nanocomposite as a new oral drug-delivery system. Materials Science and
Engineering: C. 2017;75:822-8.
[44] Gao L, Ma J, Li S, Liu D, Xu D, Cai J, et al. 2D ultrathin carbon nanosheets with
rich N/O content constructed by stripping bulk chitin for high-performance sodium
ion batteries. Nanoscale. 2019;11(26):12626-36.
[45] Lin X, Zhang L, Duan B. Polyphenol-mediated chitin self-assembly for
constructing a fully naturally resourced hydrogel with high strength and toughness.
Materials Horizons. 2021.
[46] Patel HA, Somani RS, Bajaj HC, Jasra RV. Preparation and characterization of
phosphonium montmorillonite with enhanced thermal stability. Applied Clay Science.
2007;35(3):194-200.
[47] Tyagi B, Chudasama CD, Jasra RV. Determination of structural modification in
acid activated montmorillonite clay by FT-IR spectroscopy. Spectrochimica Acta Part
A: Molecular and Biomolecular Spectroscopy. 2006;64(2):273-8.
[48] Zheng JP, Luan L, Wang HY, Xi LF, Yao KD. Study on
33
ibuprofen/montmorillonite intercalation composites as drug release system. Applied
Clay Science. 2007;36(4):297-301.
[49] Darder M, Colilla M, Ruiz-Hitzky E. Biopolymer−Clay Nanocomposites Based
on Chitosan Intercalated in Montmorillonite. Chemistry of Materials.
2003;15(20):3774-80.
[50] Mao C, Xiang Y, Liu X, Cui Z, Yang X, Yeung KWK, et al. Photo-Inspired
Antibacterial Activity and Wound Healing Acceleration by Hydrogel Embedded with
Ag/Ag@AgCl/ZnO Nanostructures. ACS Nano. 2017;11(9):9010-21.
[51] Tejiram S, Kavalukas SL, Shupp JW, Barbul A. Wound healing. 2016:3-39.
[52] Malachová K, Praus P, Pavlíčková Z, Turicová M. Activity of antibacterial
compounds immobilised on montmorillonite. Applied Clay Science.
2009;43(3):364-8.
[53] Pakyari M, arrokhi AF, Maharlooei MK, Ghahary A. Critical Role of
Transforming Growth Factor Beta in Different Phases of Wound Healing. Advances in
Wound Care. 2013;2(5):215-24.
[54] Beanes SR, Dang C, Soo C, Ting K. Skin repair and scar formation: the central
role of TGF-beta. Expert reviews in molecular medicine. 2003;5(8):1-22.
[55] Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and
regeneration. Nature. 2008;453(7193):314-21.
[56] Edwards R, Harding KG. Bacteria and wound healing. Current Opinion in
Infectious Diseases. 2004;17(2):91-6.
[57] Atiyeh BS, Hayek SN. An Update on Management of Acute and Chronic Open
Wounds: The Importance of Moist Environment in Optimal Wound Healing.
Medicinal Chemistry Reviews - Online (Discontinued). 2004.
[58] Perrault DP, Bramos A, Xu X, Shi S, Wong AK. Local Administration of
Interleukin-1 Receptor Antagonist Improves Diabetic Wound Healing. Ann Plast Surg.
2018;80(5S Suppl 5):S317-S21.
[59] Huang S-M, Wu C-S, Chiu M-H, Wu C-H, Chang Y-T, Chen G-S, et al. High
glucose environment induces M1 macrophage polarization that impairs keratinocyte
migration via TNF-α: An important mechanism to delay the diabetic wound healing.
34
Journal of Dermatological Science. 2019;96(3):159-67.
[60] Jimi S, Jaguparov A, Nurkesh A, Sultankulov B, Saparov A. Sequential Delivery
of Cryogel Released Growth Factors and Cytokines Accelerates Wound Healing and
Improves Tissue Regeneration. Frontiers in Bioengineering and Biotechnology.
2020;8.

Table 1 Reaction conditions and physical properties of quaternized chitin derivatives

Sa EPTMAC/ DQ ξ-potential, MIC (mg/mL)

mple GlcNAcU, mV E.Coli MRSA

Molar ratio

QC 5 0.26 33.96 0.20 0.20

-1

QC 10 0.34 48.47 0.15 0.15

-2

QC 15 0.48 59.57 0.10 0.05

-3

DQ: degree of quaternization;

35
Table 2. Composition and mechanical properties of sponges

Sample EW QC-3(2 wt%) MMT (5 wt%) Compressive

(g) (g) (g) Strength (MPa)

EMQA 10* 1 2 0.155

EMQS-1 10 1 2 0.056

EMQS -2 10 1 4 0.067

EMQS -3 10 1 6 0.126

36
Figure 1. (a) schematic illustration of the preparation and in-vivo application of

EMQS; (b) SEM images of (b1,b2) the composite aerogel EMQA made of

non-foaming egg white protein and (b3,b4) the composite sponge EMQS-1 made of

foaming egg white protein; (c1 and c2) the water contact angle of EMQA and

EMQS-1; (d) FTIR spectrum of freeze-dried EW protein and EW protein foam; (e, f,

g and h) mechanical properties, porosity, water uptake ability of different samples and

WVTR at different time point.

37
Figure. 2 The intercalation of QCs into MMT characterized by (a) XPS spectra, (b)

XPS peak analysis of the oxygen spectrum of QC-3, (c) XPS peak analysis of the

oxygen spectrum of QC-3 and MMT mixture; (e) XRD spectra, (f) FTIR spectra and

(g) TG profile; (h) the release efficiency of QC-3 after the incorporation of MMT.

38
Figure 3. (a) CCK-8 evaluation results of cell viability and (b, c and d) NHDF cells

culture on EMQS-2 for 48 h

39
Figure. 4 The antibacterial activity evaluation of EW, MMT and EMQS-2 and the

corresponding antibacterial rate.

40
Figure. 4 (a) The liver injury models of SD rat. The blood loss weight (b) and

bleeding time (c) of particles in the model of liver injury. *p < 0.05, **p < 0.01 and

***p < 0.001.

41
Figure. 6 (a) Macroscopic changes of the wounds covered with gauze，commercial

dressings（Tegaderm™ or Atrauman AG）and EMQS-2. (b) The wounds area treated

with gauze，commercial dressings（Tegaderm™ or Atrauman AG）and EMQS-2 at

different time point. *p < 0.05, **p < 0.01, ***p < 0.001(EMQS group compared

with gauze group). ^p < 0.05, ^^p < 0.01, ^^^p < 0.001(EMQS group compared with

commercial dressings group).

42
Figure.7 (a) Representative images of H&E staining of the wounds for the evaluation

of epidermal regeneration in wound area on days 14(original magnification, × 50)

(The black arrow shows the area without healing). (b) Representative images of H&E
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staining and Masson’s trichrome staining of the wounds (original magnification, ×

200); (c) Histopathological analysis of collagen-occupied regions by Masson’s

trichrome. NS: p > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001.

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Figure. 8 Immunohistochemical staining for CD-31 to quantify angiogenesis of the

wounds on days 14. (a) Immunohistochemical staining for CD-31 (original

magnification, × 200). (b) Histopathological analysis of CD-31+ occupied regions

CD-31 of the wounds tissue by Immunohistochemical staining. NS: p > 0.05, *p <

0.05, **p < 0.01 and ***p < 0.001.

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Figure. 9 Western-Blotting of the protein expression of growth factors (TGF-β, VEGF)

and inflammation related factors (IL-1, IL-6, IL-10 and TNF-α) and comparison of

TGF-β, VEGF, IL-1, IL-6, IL-10 and TNF-α in the wound tissue in each group on

days 7. NS: p > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001.

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Figure. 10 Secretions pathogen culture in the (a) MRSA-infected and (b)

E.Coli-infected wounds on the 3rd, 7th, 14th day; WBC in mice of MRSA-infected (c)

and (d) E.Coli-infected. *p < 0.05, **p < 0.01 and ***p < 0.001.

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Graphical Abstract